One of the major characteristics for Alzheimer's disease (AD) is deposit of amyloid plaques composed of A-beta peptides generated from cleavage of amyloid precursor protein (APP) by two secretases termed gamma and beta. Great efforts have been taken to identify compounds that inhibit the activities of these two enzymes. Recent studies have shown that BACE1, the beta-secretase undergoes alternative splicing within its exons 3 and 4 that produces isoforms with greatly reduced activity, suggesting that regulation of BACE1 splicing may be targeted for AD therapy. The goal of this proposal is to develop a cell-based assay and to use this assay to identify specific and potent small molecules that reduce A-beta generation by modulating exons 3 and 4 splicing to produce less 501-aa form of BACE1(active beta-secretase). The first aim of this project is to establish the cell-based assay for the screen. We will generate mini-gene constructs for BACE1 splicing studies with the luciferase reporter gene attached. The efficiency of alternative usage of the internal splicing sites within exons 3 and 4 of BACE1 pre-mRNA will be monitored by luciferase activities. After validation of the mini-genes in transiently transfected cells by RT-PCR, stable neuronal cell lines will be established. The second aim of this project is to use these stable cell lines to identify small molecules that reduce the production of the active isoform of 501-aa BACE1. These small molecules will be further tested for their effectiveness on reduction of A-beta generation. The investigations may lead to novel drug development for AD and understanding of signal transduction pathways regulating mRNA splicing. We will start with small scale screens using our collected compound libraries. High-throughput screens and mechanisms of action will be our long-term goals.